The present invention relates to granulocyte colony stimulating factor (xe2x80x9cG-CSFxe2x80x9d) polypeptide analog compositions, related nucleic acids, and vectors, host cells and processes for recombinant DNA production of the G-CSF analog polypeptides. More particularly the invention provides methods for screening G-CSF analogs, designed with one or more substitutions to amino acids, and selecting analogs for use as G-CSF replacements or antagonists. In addition, pharmaceutical compositions and methods of use are provided for analogs so selected.
Many therapeutic ligands elicit cellular responses by binding to cell-surface receptors to elicit cellular responses. Drug design is typically focused on the ability of a ligand to bind tightly and specifically to its intended target. However, if the drug is a protein and the target a cell-surface receptor, there are additional issues to consider from a systems-level analysis. When therapeutic ligands bind to receptors on the surface of a cell, an intracellular signaling cascade is initiated that ultimately results in an appropriate cellular response. Additionally, modulationxe2x80x94generally attenuationxe2x80x94of these signals begins almost immediately by cellular trafficking of the ligand-receptor complexes. The complexes on the surface of the cell are internalized into vesicles that fuse with endosomal compartments. From endosomes, the molecules can either be routed to degradation in lysosomes or be recycled to the cell surface intact, where free and ligand-bound receptor are redisplayed and free ligand is released to the extracellular medium. Recent evidence suggests the outcome of this sorting decision for complexes involving growth factors or cytokines often is related to the endosomal affinity constant for the ligand-receptor interaction: complexes that remain bound are readily degraded while those that dissociate are recycled [Lauffenburger et al., Chem. Biol. 5:R257-R263 (1998)]. In general, dissociation of complexes in endosomes appears to enhance receptor recycling, because it results in altered interactions between the receptors and endosomal retention components.
Additionally, for a low number of intracellular complexes, which is the case for many clinically important cytokine-receptor systems, modeling indicates a particularly strong positive correlation between the inverse endosomal affinity and the fraction of ligand recycled [French and Lauffenburger, Ann. Biomed. Eng. 25:690-707 (1997)]. Thus, if a ligand could be designed to enhance endosomal dissociation after binding to and generating signals within its target cell, the drug might reduce receptor downregulation, so that cells would be more responsive to further ligand stimulation. The lifetime and effectiveness of the drug might also be enhanced if ligand recycling were augmented by endosomal dissociation. This contrasts with the conventional approach of attempting to improve ligand potency through enhanced affinity. If extracellular affinity enhancements extend to endosomes, such attempts might actually be counterproductive because they increase receptor downregulation and possibly ligand depletion. Thus, cellular trafficking may be a bottleneck in enhancing ligand potency, particularly in cases where degradation through receptor-mediated endocytosis is significant.
A system in which the optimization of cellular trafficking properties could have a profound impact on potency is that of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR). G-CSF is a 19-kDa cytokine, which is one of the hematopoietic growth factors, also called colony stimulating factors. G-CSF is used to increase white blood cell (neutrophil) counts when blood levels of such cells are dangerously low. This commonly occurs when certain antibiotics, anti-HIV therapies and/or chemotherapies suppress the bone marrow. A recent study documents that G-CSF not only increases the number of neutrophils in the blood, but enhances the functional killing abilities of those cells as well [Vecchiarelli et al., J. Infect. Dis. 171:1448-1454 (1995)]. G-CSF specifically stimulates the proliferation and differentiation of neutrophilic precursor cells into mature neutrophils [Fukunaga et al., Cell 74:1079-1087 (1993)], and is useful for treating in neutropenic states [Welte et al., Proc. Natl. Acad. Sci. USA 82:1526-1530 (1985); Souza et al., Science 232:61-65 (1986); Gabrilove, Sem. Hematol. 26(2):1-14 (1989)]. G-CSF increases the number of circulating granulocytes and has been reported to ameliorate infection in sepsis models. G-CSF administration also inhibits the release of tumor necrosis factor (TNF), a cytokine important to tissue injury during sepsis and rejection [Wendel et al., J Immunol. 149:918-924 (1992)]. G-CSF is a member of the Group I superfamily of cytokines, characterized by an antiparallel 4-helical bundle structure and including other therapeutically important drugs such as erythropoietin and growth hormone. G-CSF binds specifically and with high affinity (apparent KDxcx9c100 pM)[Morstyn, Dexter, and Foote (eds.) Filgrastim (r-metHuG-CSF) in: Clinical Practice, Edn. 2., Marcel Dekker, Inc., New York (1998)] to G-CSFR, resulting in a ligand:receptor complex with a 2:2 stoichiometry [Horan et al., Biochemistry 35:4886-4896 (1996); Horan et al., J. Biochem.121:370-375 (1997)]. The extracellular region of G-CSFR contains the ligand-binding cytokine receptor homology (CRH) domain [Fukunaga et al., EMBO J. 10:2855-2865 (1991)] and recently, the crystal structure of G-CSF complexed with the CRH domain of G-CSFR was solved, showing the expected 2:2 ligand:receptor stoichiometry [Aritomi et al., Nature, 401:713-717 (1999)].
In humans, endogenous G-CSF is detectable in blood plasma [Jones et al., Bailliere ""s Clin. Hematol. 2(1):83-111 (1989)]. G-CSF is produced by fibroblasts, macrophages, T cells, trophoblasts, endothelial cells, and epithelial cells, and is the expression product of a single copy gene comprised of four exons and five introns located on chromosome seventeen. Transcription of this locus produces a mRNA species which is differentially processed, resulting in two forms of G-CSF mRNA, one version coding for a protein of 177 amino acids, the other coding for a protein of 174 amino acids [Nagata et al., EMBO J. 5:575-581 (1986)]. The form comprised of 174 amino acids has been found to have specific in vivo biological activity. SEQ ID NO: 1 presents a DNA encoding the 174 amino acid species of G-CSF and the corresponding sequence of amino acids is set out in SEQ ID NO: 2. G-CSF is species cross-reactive, such that when human G-CSF is administered to another mammal such as a mouse, canine, or monkey, sustained neutrophil leukocytosis is elicited [Moore et al., Proc. Natl. Acad. Sci. USA 84:7134-7138 (1987)].
Human G-CSF can be obtained and purified from a number of sources. Natural human G-CSF can be isolated from the supernatants of cultured human tumor cell lines. The development of recombinant DNA technology has enabled the production of commercial scale quantities of G-CSF in glycosylated form as a product of eukaryotic host cell expression, and of G-CSF in non-glycosylated form as a product of prokaryotic host cell expression. See, for example, U.S. Pat. No. 4,810,643 (Souza) incorporated herein by reference.
G-CSF has been found to be useful in the treatment of indications where an increase in neutrophils will provide benefits. For example, for cancer patients, G-CSF is beneficial as a means of selectively stimulating neutrophil production to compensate for hematopoietic deficits resulting from chemotherapy or radiation therapy. Other indications include treatment of various infectious diseases and related conditions, such as sepsis, which is typically caused by a metabolite of bacteria. G-CSF is also useful alone, or in combination with other compounds, such as other cytokines, for growth or expansion of cells in culture (for example, for bone marrow transplants or ex vivo expansion). G-CSF has been administered to transplant patients as an adjunct to treatment of infection or for treatment of neutropenia [Diflo et al., Hepatology 16:PA278 (1992); Wright et al., Hepatology 14:PA48 (1991); Lachaux et al., J. Ped. 123:1005-1008 (1993); Colquehoun et al., Transplantation 56:755-758 (1993)]. However, G-CSF is rapidly cleared through receptor-mediated endocytosis by peripheral neutrophils and precursor cells in bone marrow expressing G-CSFR [Morstyn, Dexter, and Foote (eds.) Filgrastim (r-metHuG-CSF) in: Clinical Practice, Edn. 2., Marcel Dekker, Inc., New York (1998)]. Thus, the potency of the drug is reduced by this negative feedback mechanism. Since cells naturally express G-CSFR in low numbers, decreasing the endosomal affinity of the complex may not only reduce receptor downregulation but may also enhance ligand recycling, as predicted by modeling [French and Lauffenburger, Ann. Biomed. Eng. 25:690-707 (1997)]. Therefore, G-CSF is a prime candidate for mutagenesis to enhance trafficking properties, thereby improving drug potency.
Various altered G-CSF""s have been reported. Generally, for design of drugs, certain changes are known to have certain structural effects. For example, deleting one cysteine could result in the unfolding of a molecule which, in its unaltered state, is normally folded via a disulfide bridge. There are other known methods to one skilled in the art for adding, deleting or substituting amino acids in order to change the function of a protein.
Recombinant human G-CSF mutants have been prepared, but the method of preparation does not include overall structure/function relationship information. For example, the mutation and biochemical modification of Cys 18 has been reported [Kuga et al., Biochem. Biophy. Res. Comm. 159:103-111 (1989); Lu et al., Arch. Biochem. Biophys. 268:81-92 (1989)].
In U.S. Pat. No. 4,810,643, entitled, xe2x80x9cProduction of Pluripotent Granulocyte Colony-Stimulating Factorxe2x80x9d (incorporated by reference herein), polypeptide analogs and peptide fragments of G-CSF are disclosed generally. Specific G-CSF analogs disclosed include those with the cysteines at positions 17, 36, 42, 64, and 74 (of the 174 amino acid species or of those having 175 amino acids, the additional amino acid being an N-terminal methionine) substituted with another amino acid (such as serine), and G-CSF with an alanine in the first (N-terminal) position.
EP 0 335 423 entitled xe2x80x9cModified human G-CSFxe2x80x9d reportedly discloses the modification of at least one amino group in a polypeptide having hG-CSF activity.
EP 0 272 703 entitled xe2x80x9cNovel Polypeptidexe2x80x9d reportedly discloses G-CSF derivatives having an amino acid substituted or deleted at or xe2x80x9cin the neighborhoodxe2x80x9d of the N-terminus. Also, Okabe et al. [Blood 75(9):1788-1793 (1990)], reportedly discloses modifications of five positions of the N-terminal region of human G-CSF.
EP 0 459 630, entitled xe2x80x9cPolypeptidesxe2x80x9d reportedly discloses derivatives of naturally occurring G-CSF having at least one of the biological properties of naturally occurring G-CSF and a solution stability of at least 35% at 5 mg/mL in which the derivative has at least Cys17 of the native sequence replaced by a Ser17 residue and Asp27 of the native sequence replaced by a Ser27 residue.
EP 0 256 843 entitled xe2x80x9cExpression of G-CSF and Muteins Thereof and Their Usesxe2x80x9d reportedly discloses a modified DNA sequence encoding G-CSF wherein the N-terminus is modified for enhanced expression of protein in recombinant host cells, without changing the amino acid sequence of the protein.
EP 0 243 153 entitled xe2x80x9cHuman G-CSF Protein Expressionxe2x80x9d reportedly discloses G-CSF to be modified by inactivating at least one yeast KEX2 protease processing site for increased yield in recombinant production using yeast.
Shaw, U.S. Pat. No. 4,904,584, entitled xe2x80x9cSite-Specific Homogeneous Modification of Polypeptidesxe2x80x9d reportedly discloses lysine altered proteins.
WO/9012874 reportedly discloses cysteine altered variants of proteins.
Australian Patent Application Document No. AU-A-10948/92, entitled, xe2x80x9cImproved Activation of Recombinant Proteinsxe2x80x9d reportedly discloses the addition of amino acids to either terminus of a G-CSF molecule for the purpose of aiding in the folding of the molecule after prokaryotic expression.
Australian Patent Application Document No. AU-A-76380/91, entitled, xe2x80x9cMuteins of the Granulocyte Colony Stimulating Factor (G-CSF)xe2x80x9d reportedly discloses muteins of G-CSF in the sequence Leu-Gly-His-Ser-Leu-Gly-Ile at position 50-56 of G-CSF with 174 amino acids, and position 53 to 59 of the G-CSF with 177 amino acids, and/or at least one of the four histidine residues at positions 43, 79, 156 and 170 of the mature G-CSF with 174 amino acids or at positions 46, 82, 159, or 173 of the mature G-CSF with 177 amino acids.
GB 2 213 821, entitled xe2x80x9cSynthetic Human Granulocyte Colony Stimulating Factor Genexe2x80x9d reportedly discloses a synthetic G-CSF-encoding nucleic acid sequence incorporating restriction sites to facilitate the cassette mutagenesis of selected regions, and flanking restriction sites to facilitate the incorporation of the gene into a desired expression system.
U.S. Pat. No. 5,214,132 reportedly discloses the modification of human G-CSF at amino acid positions 1, 3, 4, 5,and 17 [see, also, Kuga et al., Biochem. Biophys. Res. Commun. 159:103-111 (1989)].
U.S. Pat. No. 5,218,092 reportedly discloses the modification of human G-CSF at amino acid positions 1, 3, 4, 5, 17, 145, and 147.
U.S. Pat. No. 5,581,476 (incorporated by reference herein) discloses the three-dimensional structure of G-CSF to the atomic level. From this three-dimensional structure, one can forecast with substantial certainty how changes in the composition of a G-CSF molecule may result in structural changes. These structural characteristics may be correlated with biological activity to design and produce G-CSF analogs.
Signal transduction, the way in which G-CSF affects cellular metabolism, is not currently thoroughly understood. In general, G-CSF binds to and activates the G-CSF cell-surface receptor through conformational changes. This binding thereby initiates a signaling cascade (e.g., recruiting kinases to the cytoplasmic domain) which apparently initiates the changes within particular progenitor cells, leading to cellular responses such as differentiation, proliferation and migration. The G-CSF/G-CSFR complex is thought to undergo endocytic trafficking processes of internalization and sorting to recycling or degradation [Lauffenburger, and Linderman, Receptors: Models for Binding, Trafficking, and Signaling, New York: Oxford University Press (1993)].
Endocytic uptake of G-CSF potentiates intracellular proteolytic cytokine degradation in endosomal and/or lysosomal compartments. Internalized cytokine receptors if not recycled can be destroyed. Thus, endocytic trafficking could cause the depletion of G-CSF from the extracellular medium, as well as the downregulation of the G-CSF receptor. Accordingly, it would be beneficial to alter the G-CSF structure in a manner that retains proper receptor activation for signal transduction, but diminishes endocytic internalization and/or enhances endosomal sorting to recycling rather than degradation [Lauffenburger et al., Scratching The (Cell) Surface: Cytokine Engineering For Improved Ligand/Receptor Trafficking Dynamics, Chem and Biol 5:R257-R263 (1988)].
Thus, there is a need to develop better therapeutic ligands of G-CSF. Such agents would have longer half-lives and induce greater cellular proliferation if the ligand were not as prone to endocytosis and subsequent lysosomal degradation. Accordingly, it is an object of the present invention to provide these ligands and methods for producing and testing them.
The present invention provides methods of screening G-CSF amino acid substitution analogs based upon their binding affinities for the G-CSF receptor along with their ability to increase or decrease myeloid cell proliferation and thereby to assess affects on cellular trafficking and, thus, identify G-CSF analogs for use as G-CSF replacements or G-CSF antagonists.
In one aspect, therefore, the present invention relates a method for screening analogs of G-CSF for use as G-CSF replacements. This method comprises: determining the capacity of the G-CSF analog for binding to target cells and determining an equilibrium dissociation constant (Kd) for said analog; determining the capacity of the G-CSF analog for stimulating cellular proliferation (N) of target cells at a given initial ligand concentration (L); normalizing the N value obtained for the G-CSF analog with the corresponding N value for wild-type G-CSF at a given value of L to obtain Y-axis values; calculating the L/Kd values for the G-CSF analog and wild-type G-CSF to obtain X-axis values; plotting the normalized N value with the L/Kd values for the G-CSF analog and wild-type G-CSF; selecting as a G-CSF replacement, an analog displaying increased proliferation and either increased or decreased binding relative to wild-type G-CSF. As used herein, xe2x80x9cwild-type G-CSFxe2x80x9d shall mean and include the 174 amino acid species of human G-CSF set out in SEQ ID NO: 2 as well as the Metxe2x88x921 species thereof.
In another aspect, the present invention relates a method for screening analogs of G-CSF for use as G-CSF antagonists. This method comprises: determining the capacity of the G-CSF analog for binding to target cells and determining an equilibrium dissociation constant (Kd) for said analog; determining the capacity of the G-CSF analog for stimulating cellular proliferation (N) of target cells at a given initial ligand concentration (L); normalizing the N value obtained for the G-CSF analog with the corresponding N value for wild-type G-CSF at a given value of L to obtain Y-axis values; calculating the L/Kd values for the G-CSF analog and wild-type G-CSF to obtain X-axis values; plotting the normalized N value with the L/Kd values for the G-CSF analog and wild-type G-CSF; and selecting as a G-CSF antagonist an analog displaying decreased proliferation and either equal or greater binding relative to wild-type G-CSF.
Assessment of cellular proliferation, according to the invention, preferably involves determining cell number, measuring 3H-thymidine incorporation, or using an MTT assay. Furthermore, the equilibrium dissociation constant (Kd), as described herein, is preferably assessed by BIAcore analysis or ELISA.
Another aspect of the invention provides a method of treating a hematopoietic, neurological or reproduction related conditions comprised of administering an effective amount of a G-CSF replacement selected according to the method as described herein, illustrated by the exemplary analogs [Glu50]G-CSF, [Glu54]G-CSF, [Ala33]G-CSF, [Glu26]G-CSF, [Asp30]G-CSF, [Leu26]G-CSF, [Ala38]G-CSF, [Ala26]G-CSF, and the Metxe2x88x921 species thereof.
An additional aspect of the invention provides a method of treating neutrophilia comprised of administering an effective amount of a G-CSF antagonist selected according to the method as described herein, illustrated by the exemplary analog [Ala46]G-CSF, and the Metxe2x88x921 species thereof.
The invention also provides a method of treating a hematopoietic, neurological or reproduction related conditions wherein said condition is selected from the group consisting of: reduced hematopoietic function, reduced immune function, reduced neutrophil count, reduced neutrophil mobilization, mobilization of peripheral blood progenitor cells, sepsis, severe chronic neutropenia, bone marrow transplants, infectious diseases, leucopenia, thrombocytopenia, anemia, enhancing engraftment of bone marrow during transplantation, enhancing bone marrow recovery in treatment of radiation, chemical or chemotherapeutic induced bone marrow aplasia or myelosuppression, and acquired immune deficiency syndrome.
The invention further comprehends a method of sensitizing cells to chemotherapy and radiotherapy comprised of administering an effective amount of a G-CSF replacement selected according to the method as described herein.
In yet another aspect, the invention provides a method for culturing hematopoietic cells in vitro comprising: placing said cells in a suitable culture medium, said suitable culture medium containing a G-CSF replacement selected according to the method as described herein; and providing suitable conditions for the growth of said hematopoietic cells.
The invention further provides a method wherein said treatment, sensitizing, or culturing includes the use of at least one additional factor selected from among EPO, G-CSF, SCF, M-GDF, GM-CSF, M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, interleukins, IGF-1, LIF, interferon, a neurotrophic factor, flt-3/flk-2 ligand, and a fibroblast growth factor.
An additional aspect of the invention provides a kit containing components for culturing hematopoietic cells comprised of: any of the polypeptide analogs selected according to the methods as described herein; components suitable for preparing medium for culturing hematopoietic cells; and, optionally, at least one additional factor as set out above.
In yet another aspect, the target cell, as described herein, is preferably a bone marrow stem cell, a neutrophil precursor cell, an immortalized stem cell, or an acute myeloid leukemia cell.
In still another aspect, the invention provides a method of treating a hematopoietic, neurological or reproduction related conditions comprised of administering an effective amount of any of the G-CSF replacements screened according to the invention, illustratively analogs selected from the group consisting of: [Glu50]G-CSF, [Glu54]G-CSF, [Ala33]G-CSF, [Glu26]G-CSF, [Asp30]G-CSF, [Leu26]G-CSF, [Ala38]G-CSF, [Ala26]G-CSF, and the Metxe2x88x921 species thereof.
Alternately, the invention provides a method of treating neutrophilia comprised of administering an effective amount of [Ala46]G-CSF and the Metxe2x88x921 species thereof.
The invention also provides a method of sensitizing cells to chemotherapy and radiotherapy comprised of administering an effective amount of any of the G-CSF replacements screened according to the invention, illustratively analogs selected from the group consisting of: [Glu50]G-CSF, [Glu54]G-CSF, [Ala33]G-CSF, [Glu26]G-CSF, [Asp30]G-CSF, [Leu26]G-CSF, [Ala38]G-CSF, [Ala26]G-CSF, and the Metxe2x88x921 species thereof.
In another aspect, the invention provides a method for culturing hematopoietic cells in vitro comprising: placing said cells in a suitable culture medium, said suitable culture medium containing a G-CSF replacement screened according to the invention, illustratively analogs selected from the group consisting of: [Glu50]G-CSF, [Glu54]G-CSF, [Ala33]G-CSF, [Glu26]G-CSF, [Asp30]G-CSF, [Leu26]G-CSF, [Ala38]G-CSF, [Ala26]G-CSF, and the Metxe2x88x921 species thereof; and providing suitable conditions for the growth of said hematopoietic cells.
Furthermore, the invention provides a method wherein said treatment, sensitizing, or culturing with the aforementioned G-CSF analogs includes the use of at least one additional factor selected from among EPO, G-CSF, SCF, M-GDF, GM-CSF, M-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, interleukins, IGF-1, LIF, interferon, a neurotrophic factor, flt-3/flk-2 ligand, and a fibroblast growth factor.
The invention further comprehends a kit containing components for culturing hematopoietic cells comprised of: any of the G-CSF analogs screened according to the invention, illustratively analogs selected from the group consisting of: [Glu50]G-CSF, [Glu54]G-CSF, [Ala33]G-CSF, [Glu26]G-CSF, [Asp30]G-CSF, [Leu26]G-CSF, [Ala38]G-CSF, [Ala26]G-CSF, [Ala46]G-CSF, and the Metxe2x88x921 species thereof; components suitable for preparing medium for culturing hematopoietic cells; and, optionally, at least one additional factor as set out above.